A 35.7 kb DNA fragment from the Bacillus subtilis chromosome containing a putative 12.3 kb operon involved in hexuronate catabolism and a perfectly symmetrical hypothetical catabolite-responsive element.

The Bacillus subtilis strain 168 chromosomal region extending from 109 degrees to 112 degrees has been sequenced. Among the 35 ORFs identified, cotT and rapA were the only genes that had been previously mapped and sequenced. Out of ten ORFs belonging to a single putative transcription unit, seven are probably involved in hexuronate catabolism. Their sequences are homologous to Escherichia coli genes exuT, uidB, uxaA, uxaB, uxaC, uxuA and uxuB, which are all required for the uptake of free D-glucuronate, D-galacturonate and beta-glucuronide, and their transformation into glyceraldehyde 3-phosphate and pyruvate via 2-keto-3-deoxygluconate. The remaining three ORFs encode two dehydrogenases and a transcriptional regulator. The operon is preceded by a putative catabolite-responsive element (CRE), located between a hypothetical promoter and the RBS of the first gene. This element, the longest and the only so far described that is fully symmetrical, consists of a 26 bp palindrome matching the theoretical B. subtilis CRE sequence. The remaining predicted amino acid sequences that share homologies with other proteins comprise: a cytochrome P-450, a glycosyltransferase, an ATP-binding cassette transporter, a protein similar to the formate dehydrogenase alpha-subunit (FdhA), protein similar to NADH dehydrogenases, and three homologues of polypeptides that have undefined functions.

Protein-binding site at the immunoglobulin mu membrane polyadenylylation signal: possible role in transcription termination.

mRNAs specifying immunoglobulin mu and delta heavy chains are encoded by a single large, complex transcription unit (mu + delta gene). The transcriptional activity of delta gene segments in terminally differentiated, IgM-secreting B lymphocytes is 10-20 times lower than in earlier B-lineage cells expressing delta mRNA. We find that transcription of the mu + delta gene in IgM-secreting murine myeloma cells terminates within a region of 500-1000 nucleotides immediately following the mu membrane (mu m) polyadenylylation site. Transcription decreases only minimally through this region in murine cell lines representative of earlier stages in B-cell development. A DNA fragment containing the mu m polyadenylylation signal gives protein-DNA complexes with different mobilities in gel retardation assays with nuclear extracts from myeloma cells than with nuclear extracts from earlier B-lineage cells. However, using a recently developed "footprinting" procedure in which protein-DNA complexes resolved in gel retardation assays are subjected to nucleolytic cleavage while still in the polyacrylamide gel, we find that the DNA sequences protected by factors from the two cell types are indistinguishable. The factor-binding site on the DNA is located 5' of the mu m polyadenylylation signal AATAAA and includes the 15-nucleotide-long A + T-rich palindrome CTGTAAACAAATGTC. This type of palindromic binding site exhibits orientation-dependent activity consistent with the reported properties of polymerase II termination signals. This binding site is followed by two sets of directly repeated DNA sequences with different helical conformation as revealed by their reactivity with the chemical nuclease 1,10-phenanthroline-copper. The close proximity of these features to the signals for mu m mRNA processing may reflect a linkage of the processes of developmentally regulated mu m polyadenylylation and transcription termination.

How do thyroid hormone receptors bind to structurally diverse response elements?

Three classes of thyroid hormone response elements have been described. They are composed of two half-sites arranged either as a palindromic, a direct repeat or as an inverted palindromic array. Receptor homodimers as well as heterodimers can bind to all three types of response element. While the ligand binding domain of the receptors provides the major dimerization surface, asymmetric contacts between the DNA binding domains are necessary for binding to a direct repeat. Moreover, some recent findings suggest that in TR, compared to RXR, the ligand binding domain has a 180 degrees rotation with respect to the DNA binding domain. This feature could explain the preferential binding of the RXR-TR heterodimer to the direct repeat response element, in which RXR exclusively binds the 5' half-site, and of the TR homodimer to the inverted palindrome response element.

Transcriptional regulatory patterns of the myelin basic protein and malic enzyme genes by the thyroid hormone receptors alpha1 and beta1.

While there is evidence that the two ubiquitously expressed thyroid hormone (T3) receptors, TRalpha1 and TRbeta1, have distinct functional specificities, the mechanism by which they discriminate potential target genes remains largely unexplained. In this study, we demonstrate that the thyroid hormone response elements (TRE) from the malic enzyme and myelin basic protein genes (METRE and MBPTRE) respectively, are not functionally equivalent. The METRE, which is a direct repeat motif with a 4-base pair gap between the two half-site hexamers binds thyroid hormone receptor as a heterodimer with 9-cis-retinoic acid receptor (RXR) and mediates a high T3-dependent activation in response to TRalpha1 or TRbeta1 in NIH3T3 cells. In contrast, the MBPTRE, which consists of an inverted palindrome formed by two hexamers spaced by 6 base pairs, confers an efficient transactivation by TRbeta1 but a poor transactivation by TRalpha1. While both receptors form heterodimers with RXR on MBPTRE, the poor transactivation by TRalpha1 correlates also with its ability to bind efficiently as a monomer. This monomer, which is only observed with TRalpha1 bound to MBPTRE, interacts neither with N-CoR nor with SRC-1, explaining its functional inefficacy. However, in Xenopus oocytes, in which RXR proteins are not detectable, the transactivation mediated by TRalpha1 and TRbeta1 is equivalent and independent of a RXR supply, raising the question of the identity of the thyroid hormone receptor partner in these cells. Thus, in mammalian cells, the binding characteristics of TRalpha1 to MBPTRE (i.e. high monomer binding efficiency and low transactivation activity) might explain the particular pattern of T3 responsiveness of MBP gene expression during central nervous system development.